ES2659730T3 - New methanol dehydrogenase enzymes from Bacillus - Google Patents

Abstract

A nucleic acid molecule, which encodes a polypeptide that has alcohol dehydrogenase activity, in particular methanol dehydrogenase activity, that comprises or has a nucleotide sequence selected from the group consisting of: (i) a nucleotide sequence as set forth in any one of SEQ ID NO: 1 (mdh2-MGA3), 3 (mdh3-MGA3), or 5 (mdh2-PB1); (ii) a nucleotide sequence that has at least 90% sequence identity, more particularly at least 91, 92, 93 or 94% sequence identity, with a nucleotide sequence as set forth in any one of SEQ ID NO: 1, 3 or 5; (iii) a nucleotide sequence that is degenerated with any one of the nucleotide sequences of SEQ ID NO: 1, 3 or 5; (iv) a nucleotide sequence that is a part of the nucleotide sequence of any one of SEQ ID NO: 1, 3 or 5, or of a nucleotide sequence that is degenerated with a sequence of SEQ ID NO: 1, 3 or 5; (v) a nucleotide sequence encoding all or part of a polypeptide whose amino acid sequence is set forth in any one of SEQ ID NO: 2 (Mdh2-MGA3), 4 (Mdh3-MGA3) or 6 (Mdh2-PB1) ; and (vi) a nucleotide sequence encoding all or part of a polypeptide having an amino acid sequence that has at least 90% sequence identity, preferably at least 91, 92, 93 or 94% sequence identity. , with an amino acid sequence as set forth in any one of SEQ ID NO: 2, 4 or 6; or a nucleic acid molecule comprising a nucleotide sequence that is complementary to the nucleotide sequence of any one of (i) to (vi).

Description

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The term "nucleic acid molecule" as used herein refers to an RNA or DNA polymer that is single or double stranded, optionally including synthetic, unnatural or altered bases. Examples of such polynucleotides include cDNA, genomic DNA and dsRNA, inter alia. Preferably, the nucleic acid molecule is DNA. Although the nucleic acid sequences referred to herein comprise thymidine nucleotides ("t"), it will be understood that the invention also relates to the corresponding sequences in which thymidine is replaced by uridine ("u" ).

As noted above, the invention includes nucleic acid molecules that are variants of the nucleic acid molecules of SEQ ID NO: 1, 3 or 5, particularly functionally equivalent variants. The "variant" nucleic acid molecules can therefore have single or multiple nucleotide changes compared to the nucleic acid molecules of SEQ ID NO: 1, 3 or 5. For example, the variants may have 1, 2, 3, 4 or 5 or more additions, substitutions, insertions or deletions of nucleotides.

In a further aspect, the invention provides a protein (or polypeptide) having an alcohol dehydrogenase, particularly MDH activity, as defined hereinbefore.

The protein or polypeptide is preferably an MDH or a part thereof that has MDH activity. More particularly, the part maintains the function or activity of properties of the MDH from which it is derived (as defined in reference to the amino acid sequences of SEQ ID NO: 2, 4 or 6).

The protein or polypeptide can alternatively be defined in reference to the nucleic acid sequences encoding since said protein or polypeptide of the invention can be encoded by any of the nucleic acid molecules of the invention, as described above.

The invention extends to functional parts or fragments of the full-length protein molecules, meaning parts or fragments that have the same or substantially the same activity as full-length proteins as defined above, ie , functionally equivalent variants should be considered. As noted elsewhere in this document, the activity can be tested in different ways directly. Normally these functional fragments will have only small deletions with respect to the full length protein molecule, for example, of less than 50, 40, 30, 20 or 10 amino acids, although as noted above in connection with the nucleic acid molecules , greater deletions, for example, of up to 60, 70, 80, 90, 100, 150, 200 amino acids or at least 60, 70, 80, 90, 100, 150, 200 amino acids may be appropriate. In all cases, the fragments should have the same or substantially the same activity as full-length proteins as defined above, that is, they should be considered to be functionally equivalent variants. These deletions can be at the N-end, the C-end or they can be internal deletions.

Representative portions or fragments may comprise at least 50%, and preferably at least 60, 70, 75, 80, 85, 90 or 95% contiguous amino acids of the amino acid sequence as set forth in SEQ ID NO: 2 , 4 or 6.

The polypeptide of the invention as defined above therefore includes variants of the sequences of SEQ ID NO: 2, 4 or 6, for example, sequences that have certain levels of sequence identity with the exposed sequences. Such variants could be naturally occurring variants, such as comparable proteins or homologs found in other species or more particularly variants found in other microorganisms, (which share the functional properties of the encoded protein as defined elsewhere in this document. ).

Variants of naturally occurring polypeptides as defined herein can also be generated synthetically, for example, using conventional molecular biology techniques known in the art, for example, conventional mutagenesis techniques such as random mutagenesis directed to the site (for example using genetic transposition or error-prone PCR). Such mutagenesis techniques can be used to develop enzymes that have improved or different catalytic properties.

Derivatives of the polypeptides may also be used as defined herein. By derivative is meant a polypeptide as described above or a variant thereof that instead of naturally occurring amino acids, contains a structural analogue of that amino acid. The derivation or modification (for example, labeling, glycosylation, methylation of the amino acids of the protein) can also occur on condition that the function of the protein is not adversely affected.

"Structural analog" means an unconventional amino acid. Examples of such unconventional amino acids or structural analogs that can be used are amino acids D, amide isoesters (such as Nmethyl amide, retro-reverse amide, thioamide, thioester, phosphonate, ketomethylene, hydroxymethylene, fluorovinyl, (E) -vinyl, methyleneamine, methylenotium or alkane), LN methyl amino acids, D-α-methyl amino acids, DN-methyl amino acids.

Sequence identity can be evaluated by any convenient procedure. However, to determine the degree of sequence identity between sequences, computer programs that make multiple sequence alignments, for example, Clustal W (Thompson et al., (1994) Nucleic Acids Res., 22, may be useful:

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The invention further provides a microorganism or host, which may be any host microorganism as set forth above, for example, E. coli, B. subtilis and C. glutamicum, containing one or more of the nucleic acid molecules of the invention. , wherein at least one of said nucleic acid molecules is operatively linked to a heterologous expression control sequence (i.e., a heterologous expression control sequence relative to the nucleic acid molecule of the invention (), or vectors of the invention Eh host is genetically modified so that the expression of MDH is introduced or altered This can be achieved by introducing one or more copies of a nucleic acid encoding MDH of the invention under the control of a non-promoter preferably strong native, therefore the genetic material is present in the host microorganism but is not present in the microorganism of origin n atural (i.e., exogenous genetic material is present).

In general, the exogenous genetic material is introduced using the transformation procedure. The transformation normally involves a plasmid or other vector that will also contain a gene capable of identifying successfully transformed microorganisms, for example, an antibiotic resistance gene (for example against ampicillin) or some other marker. Other procedures for selecting transformants are known to the person skilled in the art and include the use of a light-sensitive vector, a lux gene, that produces positive colonies that glow in the dark. Other vehicles suitable for the transformation of bacteria include cosmids and bacteriophage molecules.

The invention will now be described further in reference to the following non-limiting Examples.

The Examples refer to the following Figures:

Figure 1: Nucleotide sequence alignment of mdh-MGA3, mdh2-MGA3, mdh3-MGA3, mdh-PB1, mdh1-PB1 and mdh2-PB1 of B. methanolicus. Figure 2: (A) Primary sequence alignments of the deduced Mdh, Mdh2 and Mdh3 proteins of B. methanolicus MGA3, and Mdh, Mdh1 and Mdh2 of PB1. (B) Primary sequence alignments of the mdh2 / mdh3 sub-family (ie, Mdh2 and Mdh3 proteins of B. methanolicus MGA3 and Mdh2 of PB1). Figure 3: Catalytic activities of Mdh (black), Mdh2 (dark gray) and Mdh3 (light gray) purified in different alcohols (200 mM) tested in vitro. (A) The substrate specificity for MDH of B. methanolicus MGA3 in vitro was analyzed. The alcoholic substrates were used at concentrations of 500 mM except for pentanol (300 mM) and hexanol (50 mM). Data were calculated from the average value of two experiments that were done in triplicate. (B) The substrate specificity for MDH of B. methanolicus PB1 was analyzed in vitro. The alcoholic substrates were used at concentrations of 500 mM except for pentanol (300 mM) and hexanol (50 mM). Data were calculated from the average value of two experiments that were done in triplicate. Figure 4: (A) Determination of the optimum temperature for the catalytic activity of Mdh, Mdh2 and Mdh3 in vitro.

(B) The determination of the optimum temperature conditions for catalysis by the MDH proteins of B. methanolicus PB1 was carried out in vitro: the specific activity was calculated in 500 mM ethanol; the measurements were made in triplicate. Figure 5: (A) The stability temperature of MGA3 Mdh, Mdh2 and Mdh3 was tested in vitro. The enzymes were incubated at 45 ° C or at 60 ° C before the enzymatic assay. (B) The stability temperature for Mdh, Mdh1 and Mdh2 of PB1 was tested in vitro. The enzymes were incubated at 45 ° C or at 60 ° C before the enzymatic assay. Figure 6: The catalytic activity of Mdh, Mdh2 and Mdh3 was tested in the presence of Act and compared with the level of activity measured when Act was not present. (A) The activation of MDH with Act of B. methanolicus MGA3 was tested in vitro. The assays were carried out in triplicate with 500 mM of alcohol and 5 µg / ml of MDH and Act proteins. (B) Activation of the MDH with Act of B. methanolicus PB1 in vitro. Triplicate assays were carried out with 500 mM of alcohol and 5 µg / ml of MDH and Act proteins. Figure 7. (A) Cloning strategies. (B) Physical map of the act-pHCMC04 plasmid. Figure 8. In vitro activities of recombinant strains of B. subtilis when tested using ethanol and methanol as substrates. Figure 9. Mass% of the isotopomeric fraction M1 of different metabolites before (i.e., the zero moment) and after (i.e. at time points of 30 and 90 minutes) of the addition of 13C-methanol. The three lines represent the results of three independent biological repeats. A: C. glutamicum delta ald pEKEX3; B: C. glutamicum delta ald strain expressing Mdh2 (pVWEx1-Mdh2), Hps and Phi (pEKEX3 -Hps + Phi). PEP: phosphoenolpyruvate; 2/3 P: 2-and 3-phosphoglycerate; FBP: fructose bis-phosphate; R5P: ribose-5-phosphate. Figure 10: Metabolic labeling using 13C-methanol or 13C-formaldehyde as substrate. (A) ΔfrmA cells expressing mdh2 and hps phi with 13C-methanol as a carbon source. (B) ΔfrmA cells expressing hps and phi with 13C-formaldehyde as a carbon source. Figure 11: Construction of the synthetic operon. Each consecutive gene is introduced at the SwaI / BglII restriction sites. The last gene in the operon contains the His6 marker. For experiments with 13C labeling (see Example 18), plasmids AMAhxlB- and AMABGFTPrpe-pHCMC04 were used. RBS: ribosome binding site. Figure 12: SDS-PAGE of the purified proteins expressed in B. subtilis 168. Strains of B. subtilis containing any one of the constructs shown in Figure 11 were used. The proteins were purified using a HisTrap column and concentrated. using Vivaspin columns. Protein bands are indicated in tables. M: molecular weight marker.

Figure 13:% of mass of the isotopomeric fraction M1 of different metabolites (ie at the zero moment) and then (that is, at the time points of 30 and 90 minutes) of the addition of 13C-methanol. The two lines represent the results of two independent biological repeats. (A): B. subtilis 168 pHCMC04; (B): B. subtilis 168 AM3AhxlB-pHCMC04; (C): B. subtilis 168 AM3ABGFTPrpe-pHCMC04. PEP: phosphoenolpyruvate; 2/3 PG: 2-and 3-phosphoglycerate; FBP: fructose-bis-phosphate.

Examples

Table 1: Bacterial strains and plasmids used in the present study

Oplásmido strain

Description Reference (s) or source

B. methanolicus MGA3

ATCC53907 wild type strain ATCC

B. methanolicus PB1

ATCC wild type strain ATCC

E. coli DH5α

General Cloning Guest Bethesda Research Laboratories

E. coli ER2566

It houses the chromosomal gene for T7 RNA polymerase New England Biolabs

pHP13

E. coli-B shuttle vector. methanolicus, Cmr (Haima, Bron et al. (1987) Mol Gen Genet 209 (2): 335-342; Jakobsen, Benichou et al. (2006) J Bacteriol 188 (8): 30633072)

pGEM-T

E. coli cloning vector; Ampr Promise

pLITMUS28

E. coli cloning vector; Ampr Promise

pET21a

E. coli expression vector, six-His marker, T7 promoter, Ampr Novagen

pTMB1

pLITMUS28 with the mdh2 gene of MGA3 This studio

pTMB2

pLITMUS28 with the mdh3 gene of MGA3 This studio

pET21a_MGA3mdh

pET21a with the mdh coding region of MGA3 under the control of T7 and fused to the six-His marker This studio

pET21a_MGA3mdh2

pET21a with the mdh2 coding region of MGA3 under the control of T7 and fused to the six-His marker This studio

pET21a_MGA3mdh3

pET21a with the mdh3 coding region of MGA3 under the control of T7 and fused to the six-His marker This studio

pET21a_MGA3act

pET21a with the MGA3 act coding region under the control of T7 and fused to the six-His marker This studio

pET21a_PB1mdh

pET21a with the mdh coding region of PB1 under the control of T7 and fused to the six-His marker This studio

pET21a_PB1mdh1

pET21a with the mdh1 coding region of PB1 under the control of T7 and fused to the six-His marker This studio

pET21a_PB1mdh2

pET21a with the mdh2 coding region of PB1 under the control of T7 and fused to the six-His marker This studio

pET21a_PB1act

pET21a with the act coding region of PB1 under the control of T7 and fused to the six-His marker This studio

pET21a-nudF

pET21a with the nudF coding region of B. subtilis under the control of T7 and fused to the six-His marker This studio

B. subtilis 168

Wild type strain 168 Kunst et al. (1997) Nature 390: 249-256

(continuation)

Oplásmido strain

Description Reference (s) or source

pHB201

Shuttle vector E. coli-B. Subtilis, Cmr, Emr Bron et al. (1998) J Biotech 64: 3-13

pHCMC04

Shuttle vector E. coli-B. subtilis, Cmr Nguyen et al. (2005) Plasmid 54: 241-248

act-pHCMC04

pHCMC04 with the act coding gene of B. methanolicus MGA3 under the control of the xylose inducible promoter and fused to the six-His marker This studio

mdh-pHCMC04

pHCMC04 with the mdh coding gene of B. methanolicus MGA3 under the control of the xylose inducible promoter and fused to the six-His marker This studio

mdh2pHCMC04

pHCMC04 with the mdh2 coding gene of B. methanolicus MGA3 under the control of the xylose inducible promoter and fused to the six-His marker This studio

mdh3pHCMC04

pHCMC04 with the mdh3 coding gene of B. methanolicus MGA3 under the control of the xylose inducible promoter and fused to the six-His marker This studio

AmdhpHCMC04

pHCMC04 with the coding act and mdh genes of B. methanolicus MGA3 under the control of the xylose and mdh inducible promoter fused to the six-His marker This studio

Amdh2pHCMC04

pHCMC04 with the coding act and mdh2 genes of B. methanolicus MGA3 under the control of the xylose and mdh2 inducible promoter fused to the six-His marker This studio

Amdh3pHCMC04

pHCMC04 with the coding act and mdh3 genes of B. methanolicus MGA3 under the control of the xylose and mdh3 inducible promoter fused to the six-His marker This studio

Ampr, ampicillin resistance; Cmr, chloramphenicol resistance

Materials and procedures

Biological materials, DNA modification, and culture conditions

5 The bacterial strains and plasmids used in this study have been listed in Table 1. E. coli DH5α was used as a conventional cloning host while E. coli ER2566 was used as a host for recombinant protein expression MDH, Act and NudF. E. coli strains were generally grown at 37 ° C in liquid or solid Luria-Bertani (LB) medium (Sambrook (2001) Cold Spring Harbor Laboratory Press) supplemented with ampicillin (100 µg / ml) or chloramphenicol (10 µg / ml) when appropriate. The procedures in E. coli

10 recombinants were carried out as described by Sambrook and Russell (2001; Cold Spring Harbor Laboratory Press). PCR were carried out using the Expand High Fidelity PCR system (Roche Applied Science, Indianapolis, IN) and DNA sequencing was carried out by Eurofins MWG Operon (Ebersberg, Germany, www.eurofinsd-na.com). The isolation of the total DNA of B. methanolicus MGA3 and PB1 and the recombinant production of MDH, Act and NudF proteins in E. coli ER2566 was carried out as described previously (Brautaset and

15 col., (2004) J Bacteriol 186 (5): 1229-1238; Brautaset et al., (2010) Appl Microbiol Biotechnol 87 (3): 951-964). The transformation of B. methanolicus MGA3 was carried out by electroporation Jakobsen et al. (2006) J Bacteriol 188 (8): 3063-3072). B. methanolicus cells were grown at 50 ° C in 100 ml of MeOH200 medium, containing 200 mM methanol, in Mann10 medium containing 10 g / liter of mannitol, or SOBsuc medium (Jakobsen et al. (2006) J Bacteriol 188 (8): 3063-3072), and chloramphenicol (5 µg / ml) was added if appropriate.

20 Construction of expression vectors

pET21a_mdh-MGA3, pET21a_mdh2-MGA3, pET21a_mdh3-MGA3 and pET21a_act-MGA3:

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Due to the high sequence similarity between the mdh2 and mdh3 coding regions of MGA3, primers were designed for PCR amplification and concomitant cloning based on unique sequences representing the regions surrounding the respective genes, and are as follows:

con16_rev: 5’-AACCATGGATGAGGAGGATGTTTGTATGAC-3 ’(SEQ ID NO: 13) and con18_rev: 5’-AACCATGGCAAACAAAGGGGATGTATGTATG-3’ (SEQ ID NO: 14); con41_rev: 5’-AGGATCCCCTCCGTTTTGTCGTATTAC-3 ’(SEQ ID NO: 15) and con43_rev: 5’-TGGATCCTCTTCGTCTTTGGCGAATTAC-3’ (SEQ ID NO: 16).

The respective DNA fragments were digested with NcoI + BamHI (recognition sites are underlined in the primer sequences), and ligated into the corresponding sites of pLITMUS28 resulting in the plasmid, pTMB1 that hosts mdh2 and pTMB2 that hosts mdh3. The MDH genes cloned in both plasmids were then sequenced. Next, the coding regions of mdh and act were amplified by PCR from the total DNA of B. methanolicus MGA3, and the coding regions of mdh2 and mdh3 were amplified by PCR from plasmids pTMB1 and pTMB2, respectively, using the following PCR primer pairs:

mdh_fwd-MGA3: 5’-CATATGACAACAAACTTTTTCATTCC-3 ’(SEQ ID NO: 17) and mdh_rev-MGA3: 5’-CTCGAGCATAGCGTTTTTGATGATTTGTG-3’ (SEQ ID NO: 18); mdh2_fwd-MGA3: 5’-CATATGACAAACACTCAAAGTGC-3 ’(SEQ ID NO: 19) and mdh2_rev-MGA3: 5’-CTCGAGCATCGCATTTTTAATAATTTGG-3’ (SEQ ID NO: 20); mdh3_fwd-MGA3: 5’-CATATGAAAAACACTCAAAGTGCATTTTAC-3 ’(SEQ ID NO: 21) and mdh_rev-MGA3: 5’-CTCGAGCATAGCGTTTTTGATGATTTGTG-3’ (SEQ ID NO: 22); act_fwd-MGA3: 5’-AAACATATGGGAAAATTATTTGAGG-3 ’(SEQ ID NO: 23) and act_rev-MGA3: 5’-AAACTCGAGTTTATTTTTGAGAGCCTCTTG-3’ (SEQ ID NO: 24);

The restriction sites for NdeI and XhoI, respectively, are underlined in the direct and inverse primers. The resulting PCR products mdh-MGA3 (1149 bp), mdh2-MGA3 (1163 bp), mdh3-MGA3 (1165 bp) and act-MGA3 (570 bp) joined A / T directly into the general cloning vector pGEM -T, and the respective cloned inserts were verified by DNA sequencing. The resulting vectors were then digested with XhoI and NdeI and the inserts were ligated at the corresponding sites in phase with the sequence of the six-His marker of plasmid pET21a to give rise to plasmids pET21a_mdh-MGA3, pET21a_mdh2-MGA3, pET21a_mdh3-MGA3 , and pET21a_act-MGA3, respectively.

pET21a_mdh-PB1, pET21a_mdh1-PB1, pET21a_mdh2-PB1, and pET21a_act-PB1:

The coding regions of the mdh-PB1, mdh1-PB1 and mdh2-PB1 genes were amplified by PCR from the total DNA of PB1 using the following primer pairs:

mdh_fwd-PB1: 5’-ATACATATGACGCAAAGAAACTTTTTCATTC-3 ’(SEQ ID NO: 25) and mdh_rev-PB1: 5’-ATACTCGAGCAGAGCGTTTTTGATGATTTG-3’ (SEQ ID NO: 26); mdh1_fwd-PB1: 5’-ATACATATGACTAAAACAAAATTTTTCATTC-3 ’(SEQ ID NO: 27) and mdh_rev-PB1 (see above); mdh2_fwd-PB1: 5’-ATACATATGACAAACACTCAAAGTATATTTTAC-3 ’(SEQ ID NO: 28) and mdh2_rev-PB1: 5’-ATACTCGAGCATAGCATTTTTAATAATTTGTATAAC-3’ (SEQ ID NO: 29)

The three products of the resulting PCR mdh-PB1 (1164 bp), mdh1-PB1 (1164 bp) and mdh2-PB1 (1170 bp) were ligated A / T into plasmid pGEM-T. The resulting plasmids were digested with XhoI and NdeI (restriction sites are underlined in the primers) and ligated into the corresponding sites of plasmid pET21a, giving rise to plasmids pET21a_mdh-PB1, pET21a_mdh1-PB1, and pET21a_mdh2-PB1, respectively . The act-PB1 coding region was amplified from total PB1 DNA using the pair of primers:

act_frw-PB1: 5’-TTTTCATATGGGAAAATTATTTGAGGAAA-3 ’(SEQ ID NO: 30) and act_rev-PB1: 5’-TTTTCTCGAGTTTATTTTTGAGAGCCTCTTG-3’ (SEQ ID NO: 31).

The act-PB1 PCR product was digested with NdeI and XhoI (restriction sites are underlined in the primers) and ligated into the corresponding sites of pET21a, resulting in plasmid pET21a_act-PB1.

pET21a_nudF:

The nudF coding region was amplified by PCR from the total B. subtilis DNA using the following pair of primers:

nudF-fwd: 5’-TTTTCATATGAAATCATTAGAAGAAAAAACAATTG-3 ’(SEQ ID NO: 32) and nudF-rev: 5’-TTTTCTCGAGTTTTTGTGCTTGGAGCGCTT-3’ (SEQ ID NO: 33).

The resulting PCR product (572 bp) was ligated A / T into pGEM-T and the cloned insert was verified by DNA sequencing. The resulting vector was digested with XhoI and NdeI (restriction sites are underlined in

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mdh2-MGA3 fwd: 5’-GGATACATGTCAAACACTCAAAGTGC-3 ’(SEQ ID NO: 36) and

mdh2-MGA3 rev: 5’-TCTAGACACCATCGCATTTTTAATAATTTGG-3 ’(SEQ ID NO: 37);

mdh3-MGA3 fwd: 5’-GGATACATGTAAAACACTCAAAGTGC-3 ’(SEQ ID NO: 38) and

mdh3-MGA3 rev: 5’-TCTAGACACCATAGCATTTTTAATAATTTGGATG-3 ’(SEQ ID NO: 39);

mdh-PB1 fwd: 5’-TCCACCAGCTAGCGTAATTGG-3 ’(SEQ ID NO: 40) and

mdh-PB1 rev: 5’-AACCTGTGCCATGAAGAAATGC-3 ’(SEQ ID NO: 41);

mdh1-PB1 fwd: 5’-TCCATCATCCACTGTATTTGG-3 ’(SEQ ID NO: 42) and

mdh1-PB1 rev: 5’-ACCTGTGCTGTGAAGGAATGC-3 ’(SEQ ID NO: 43);

mdh2-PB1 fwd: 5’-CGTGAAGCTGGTGTGGAAGTATT-3 ’(SEQ ID NO: 44) and

mdh2-PB1 rev: 5’-TCCAAACCTTCTGCGACGTT-3 ’(SEQ ID NO: 45).

With respect to the quantification of the genes in question, it was carried out by normalizing the results, with respect to 16s RNA (endogenous control) and a calibration sample, using a comparative Ct procedure (2-ΔΔCt procedure) as previously described ( Heid, Stevens et al. (1996) Genome Res 6 (10): 986994; Jakobsen, Benichou et al. (2006) J Bacteriol 188 (8): 3063-3072; Brautaset, Jakobsen et al. (2010) Appl Microbiol Biotechnol 87 (3): 951-964). The relative differences in the levels of transcription of the three genes were determined by calculating the ΔCT values given as follows: mdh2 (Ct mdh2-Ct mdh) and the ΔCT value of mdh3 (Ct mdh3-Ct mdh). The efficiency of the primer of the three genes was tested before the other experiments were carried out.

3D model of deduced MDH proteins

Structural models of Mdh and Mdh2 proteins of MGA3 were made using the fully automatic protein structural model-homology server SWISS-MODEL (http://swissmodel.expasy.org/) (Peitsch (1995) Bio-Technology 13 (7): 658-660; Arnold, Bordoli et al. (2006) Bioinformatics 22 (2): 195-201; Kiefer, Arnold et al. (2009) Nucleic Acids Res 37 (Database issue): D387-392). Due to the high homology between the primary structures deduced from the Mdh2 and Mdh3 proteins of MGA3, no model search for Mdh3 was performed. Blast searches with gaps (Altschul et al (1997) J Mol Biol 215 (3): 403-410; Schäffer et al. (2001) Nucleic Acids Res. 29 (14): 2994-3005) resulted in 9 successes of Common matrix with E values ranging from 1 · e-98 (pdb: 3 bfj, 1,3-propanediol oxidoreductase) to 1 · e-17 (pdb: 1oj7, E. coli K12 YQHD) for Mdh and from 1 · e-112 (pdb: 3bfj) up to 2 · e-14 (pdb: 1oj7) for Mdh2. The 3D alignments of the matrix files using Deep view / Swiss pdb viewer (Guex and Peitsch (1997) Electrophoresis 18 (15): 2714-2723) showed that they had all very similar folds and structural models based on the 3 bfj matrix, Having the highest amino acid similarity score for both Mdh and Mdh2, they were chosen to represent Mdh and Mdh2. The Deep view / Swiss PDB viewer was also used to visualize the structural models of the MDH.

Example 1: Genetic organization of methanol dehydrogenases and protein activating genes in strains of wild type B. methanolicus MGA3 and PB1

The in silico exploration of the genomic sequence of B. methanolicus MGA3 (Heggeset et al., 2011) identified mdh encoded by plasmid pBM19, denoted herein as mdh-MGA3, and two more supposed MDH coding genes in the genome of MGA3, denoted herein as mdh2-MGA3 and mdh3-MGA3, located remotely on the chromosome. The coding sequences mdh2-MGA3 and mdh3-MGA3 were 96% identical to each other and 65% and 66% identical, respectively to the coding sequence mdh-MGA3. Alignment of the primary sequences of the deduced polypeptides Mdh2-MGA3 and Mdh3-MGA3 revealed that they were 96% identical to each other, and 61% and 62% identical, respectively, to Mdh-MGA3 (Figure 2).

The inventors have obtained semi-continuous fermentation results in methanol that demonstrate that the two strains of wild-type B. methanolicus MGA3 and PB1 are substantially different with respect to methylotrophic properties. Inspection of the genomic sequence of PB1 confirms the presence of three genes encoding different MDH and one act gene, analogous to MGA3. The mdh-PB1 gene that is located in plasmid pBM20 was 92% identical to the mdh-MGA3 gene of MGA3 and the respective genetic products had 93% primary sequence identity (Figure 2). In contrast to MGA3, the sequences of the two chromosomal genes of PB1, denoted as mdh1-PB1 and mdh2-PB1, were not very similar. The mdh1-PB1 gene encodes an alleged Mdh1 protein with 92% primary sequence identity with the Mdh Protein of MGA3 while the mdh2-PB1 encodes an alleged Mdh2 protein with 91% and 92% primary sequence identity with respect to Mdh2 and Mdh3 proteins of MGA3, respectively. Based on these sequence analyzes, it seems as if MGA3 and PB1 possess two subtypes of MDH coding genes; the type "mdh / mdh1" and the type "mdh2 / mdh3". The MGA3 strain has only one mdh / mdh1 type gene (pBM19) and two mdh2 / mdh3 type genes (chromosome), while strain PB1 has two mdh / mdh1 type genes (pBM20 and chromosome) and one mdh / mdh3 type gene ( chromosome). The biological impact of these different was investigated further later.

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Example 12: MDHs in general have higher Vmax values and lower Km values for formaldehyde compared to methanol

The biological significance of MDH for the oxidation of methanol during methylotrophic growth is not ambiguous, while the biological role of this enzyme as a formaldehyde detoxification system in cells that consume methanol is less investigated. It was shown here that all enzymes exhibited both formaldehyde and acetaldehyde reductase activities (see above), and the inventors chose to characterize this property kinetically. Using formaldehyde as substrate, the Km values are 1 mM, 5 mM and 15 mM and the corresponding Vmax values were 1.4 U / mg, 1.5 U / mg and 5 U / mg for the Mdh, Mdh2 and Mdh3 of MGA3, respectively (Table 2). For PB1 proteins, the Km values for formaldehyde were 2.5 mM, 4 mM and 1.3 mM, respectively, and the corresponding Vmax values were 0.45 U / mg, 0.53 U / mg and 1 , 12 U / mg, respectively. Together these results show that the six MDHs in general have a higher affinity and a higher Vmax when formaldehyde is the substrate, compared to when methanol is the substrate.

Example 13: The three mdh genes are transcribed at different levels in exponentially growing B. methanolicus cells.

It had previously been shown that mdh-MGA3 transcription is presumably very high in B. methanolicus cells and slightly positively regulated (approximately 3 times) in cells grown on methanol versus mannitol, while act transcription levels were similar in both growth conditions (Jakobsen et al., (2006) J Bacteriol 188 (8): 3063-3072). Here, the three MDHs encoding the MGA3 genes were included in similar analyzes and the results showed that the relative transcription levels of mdh-MGA3 and mdh2-MGA3 were 2 times and 3 times higher in methanol compared to mannitol. The level of transcription of mdh3-MGA3 was essentially similar in the two different culture conditions. For mdh this result was somewhat different compared to previous data (Jakobsen et al. (2006) J Bacteriol 188 (8): 3063-3072), and the reason for this is unknown. Interestingly, the Ct values obtained under conventional conditions for the three genes were highly different, with mdh-MGA3 representing by far the lowest values indicating higher transcription levels. It was found that Ct mdh2-Ct mdh was 8 and Ct mdh3-Ct mdh was 14 (taking into account a primer efficiency of approximately 100% in all these experiments, these numbers should imply that the transcription levels mdh2-MGA3 and mdh3 -MGA3 were approximately 250 times and 10,000 times lower than the level of mdh transcription, respectively, under the conditions tested).

The coding sequences mdh2-MGA3 and mdh3-MGA3 are 96% identical at the DNA level and to rule out any cross hybridization of the rt-PCR primers in these experiments, the respective pairs of rt-PCR primers (see Materials and procedures) were tested for plasmid DNAs, pTMB1 and pTMB2, which harbored the respective genetic sequences of mdh2 and mdh3. The results clearly show that no PCR product was obtained when mdh2 specific primers were used together with pTMB2 DNA, or alternatively when mdh3 specific primers were used together with the pTMB1 DNA matrix (data not shown). These data confirmed that the rt-PCR primers used for mdh2-MGA3 and mdh3-MGA3 are specific for their specific targets, confirming that the data obtained should be reliable.

A similar analysis was carried out for the mdh of PB1 and the results showed that the transcription levels of mdh-PB1 and mdh2-PB1 were essentially similar in the mannitol culture versus methanol. Surprisingly, the level of transcription of mdh1-PB1 in mannitol was 14 times higher than in methanol, and the biological impact of this remained unknown. As for MGA3, the inventors realized that the relative transcription level of these three genes in PB1 was presumably very different, and the mdh-PB1 gene was transcribed at levels much higher than mdh1-PB1 and mdh2-PB1 (data not shown).

Example 14: Expression of mdh genes from B. methanolicus in E. coli

Construction of expression vectors

The genes coding for Mdh and the Mdh activating Act protein were amplified from plasmids pET21a that harbored genes from strains MGA3 (mdh-MGA3, mdh2-MGA3, mdh3-MGA3 and act-MGA3) and PB1 (mdh-PB1, mdh1-PB1 and mdh2-PB1) from B. methanolicus. The genes were then cloned into plasmid pSEVA424 (mdh genes) or plasmid pSEVA131 (act gene). For the cloning of mdh-MGA3, mdh-PB1, mdh1-PB1 and act-MGA3, EcoRI and HindIII restriction sites were used, while mdh2-MGA3, mdh2-PB1 and mdh3-MGA3 were cloned using restriction sites EcoRI and PstI. The resulting restriction vectors were transformed into electrocompetent E. coli K12 (BW25113) ts and E. coli K-12 (BW25113) with a frmA gene removed.

Expression experiments

For expression experiments, the cells were cultured in Luria-Bertani medium (LB) for in vitro assays or in M9 medium for in vivo assays, both containing 20 µg / ml streptomycin for pSEVA424. When Act was co-expressed, the medium was supplemented with 100 µg / ml ampicillin. Expression was induced when the cells reached an OD of 0.5 (for in vitro assays) or OD 1 (for in vivo assays) by adding 0.1 mM of IPTG

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For the construction of vectors for the co-expression of act and the three different methanol dehydrogenase genes, the methanol dehydrogenase genes were amplified by PCR with a direct primer containing a stop codon and the RBS mntA of B. subtilis and a reverse primer containing a short crimp containing the restriction sites SwaI and BgIII (Figure 7B). The respective genes were then digested at the end with StuI and BgIII and ligated into the SwaI and BgIII sites of the act-pHB201 vector. In this way, a stop codon is introduced after the act gene and the methanol dehydrogenase genes now contain the His6 marker. After sequencing the genes were transferred to plasmid pHCMC04 (Figure 7B) using the SpeI and BamHI restriction sites. Insertions were confirmed by sequencing.

Establishment of recombinant B. subtilis cells expressing methanol dehydrogenase

168 cells of B. subtilis were transformed with the expression plasmids act-MGA3, mdh-MGA3, mdh2-MGA3 and mdh3-MGA3, and the analogous vectors that co-expressed each of the methanol dehydrogenase genes with the gene act- MGA3. Positive colonies were collected from the plate and the plasmid was isolated and positive clones were checked by restriction. The colonies were collected from the plate and grown overnight at 37 ° C (250 rpm) and diluted to an OD 600 = 0.1 in 20 ml of LB containing chloramphenicol (5 µg / ml). After 3 h of culture at 37 ° C (250 rpm), 500 µl of 40% xylose was added to the culture to induce expression. The culture was cultured for another 3 h and 2 ml samples were taken. The sample was centrifuged for 2 min, 11,000 x g and the supernatant was removed. The agglomerate was re-suspended in 300 µl of Birnboim A for B. subtilis (10 mM Tris-HCl pH 8.0; 20% sucrose; 50 mM NaCl; 0.25 mg / ml lysozyme; inhibitor protease) and incubated at 37 ° C for 30 minutes. Samples were stored at -80 ° C before use. In addition, the inventors took advantage of recombinant E. coli cells constructed in the project that express mdh-MGA3, mdh2-MGA3, mdh3-MGA3 and act-MGA3 from plasmid pET21a in the in vitro assays.

In vitro assays for methanol dehydrogenase activity

The activities of the Mdh proteins were measured following the formation of NADH spectrophotometrically. The reaction mixture contained:

-100 µl Glycine-KOH (pH 9.5) -100 µl of 5 M Methanol (or 5 M ethanol) -5 µlMgSO41 M -10 µl of NAD + 50 mM -10 µl of sample - (10 µl of E. lysate) coli act-pET21a) -775 µl H2O

The cuvettes and reaction mixture without cell lysate were pre-incubated at 50 ° C. NADH formation was followed for 10 minutes at 340 nm at 50 ° C. The total activity was calculated by dividing the increase in absorption units per minute by the extinction coefficient (6.23 cm-1 mm-1) and the total protein concentration in U / mg of total protein. All tests were carried out using both methanol and ethanol as an alternative substrate.

Results

The three genes tested, mdh-MGA3, mdh2-MGA3, and mdh3-MGA3 expressed active methanol dehydrogenases in host B. subtilis, while act-MGA3 alone does not express detectable methanol dehydrogenase activity (Figure 8). In general, the activities were significantly higher when ethanol is used instead of methanol as a substrate, for the three genes tested (this is similar to those observed when these enzymes have been purified from recombinant E. coli cells). In all cases, methanol dehydrogenase activities were significantly stimulated by Act. When the act and methanol dehydrogenase genes were coexpressed from the same plasmid in B. subtilis 168, it appears that the Mdh2 protein is the most active. However, the inventors realized that when Act is supplied from the E. coli lysates, then the mdh-MGA3 gene is the most active. Therefore, the mdh2-MGA3 gene - co-expressed together with act-MGA3 - is in total the best choice for maximized methanol dehydrogenase activity in B. subtilis when tested in vitro.

Example 16: Incorporation of methanol in genetically modified C. glutamicum

13C marking experiments

For the labeling experiments the inventors used the delta ald strain of C. glutamicum expressing Mdh2-MGA3 (pVWEx1-Mdh2), Hps and Phi (pEKEX3 -Hps + Phi). As a negative control, the alta strain of C. glutamicum with the empty plasmid pEKEX3 was used. All strains of C. glutamicum were grown in M8 medium containing (per liter) 3.48 g of Na2HPO412 H20, 0.60 g of KH2PO4, 0.51 g of NaCl, 2.04 g of NH4Cl, 0 , 10 g of MgSO4, 4.38 mg of CaCl2, 15 mg of Na2EDTA · 2 H2O, 4.5 mg of ZnSO4 · 7 H2O, 0.3 mg of CoCl2 · 6 H2O, 1 mg of MnCl2 · 4 H2O, 1 mg of H3BO3, 0.4 mg of Na2MoO4 · 2 H2O, 3 mg of FeSO4 • 7 H2O and 0.3 mg of CuSO4 · 5 H2O. For all cultures (M9 or LB), 1 mM of IPTG was used as inducer and 100 µg / ml of spectinomycin and 25 µg / ml of kanamycin were added in the medium as resistance markers. All cultures were carried out at 30 ° C. Cell strains from glycerol were plated as raw material in a

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Claims (4)

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13.

The host microorganism of claim 12, wherein the host microorganism is a bacterium selected from the genus Escherichia, Corynebacterium or Bacillus.

14.

 The host microorganism of claim 12 or claim 13, wherein the host microorganism is E. coli, C. glutamicum, B. subtilis or B. methanolicus.

fifteen.

A method for the introduction or modification of the alcohol dehydrogenase activity, and in particular MDH activity, in a host microorganism, said method comprising the introduction into said microorganism of a nucleic acid molecule as defined in any one of claims 1 to 5 and the cultivation of said microorganism under conditions in which said nucleic acid molecule is expressed.

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